HEAVY METALS TOXICITY IN ANIMALS

Dr. V.K. GUPTASenior Scientist

Division of MedicineIndian Veterinary Research Institute,

Izatnagar,

Itai-itai disease :Toyama, Japan, starting around 1912

Mitsui Mining & Smelting Co., Ltd, Jinzu river

Minamata (1953--60) & Niigata(1964--65) disease in Japan

Chandigarh in early 1978, West Bengal in 1984

AREA AFFECTED WITH ARSENIC

Fifty districts of Bangladesh and 9 districts in West Bengal, India have arsenic levels in groundwater above the maximum permissible limit of 50 μg/L . ( WHO )

ARSENIC TOXICITY AREA IN WEST BENGAL

CITITES AT RISK IN INDIALEAD CONTENT IN WATER

Alarming High Medium Low

Kolkata Delhi Chennai Bengaluru

Kochi Coimbatore Ludhiana Ahmedabad

Mumbai Madurai Surat Hyderabad

Pune Bhubaneswar Ghaziabad Indore

Nagpur   Jamshedpur Bhopal

Nashik     Chandigarh

Guwahati     Lucknow

      Mangalore

      Mysore

Pb Hg Cd As Ratlam (M.P.)

Kodaikanal (T.N.)

Kamrup,Dhemaji(Assam)

Tuticorin(T.N.)

Bandalamottu Mines (A.P.)

Ganjam (orissa)

Pathanamthitta (Kerala)

West Bengal

Vadodara (Gujrat)

Singrauli (M.P.)

Ballia (U.P.)

Korba (Chattisgarh)

HEAVY METALS CONTAMINATED AREA IN INDIA

Gautam SP, CPCB, New Delhi, Ram Murty, Indian Institute of Toxilogy Research Buragohain et al.,2013, Shakhila et al.,2014

INTRODUCTION

All living creatures requires minerals

Naturally metals are distributed in environment during earth's origin.

Rapid industrialization Overgrowing urbanization Environmental manipulation (Jarup L, 2003; Waldron and Ediing,1997)

Threshold level

Deficiency disease

Normal healthy life

Toxicity

Heavy Metals

Non essentialBa, Li, Zr

Less toxicSn, Al

Highly toxicPb, Hg, Cd

Essential Cu, Zn, Co, Cr, Mn, Fe

On health effects basis

Metal having atomic weight greater than sodium (23) and specific gravity (density) > 5gm/cm3 (Hollemen and Wiverd,1985)

(Mukesh K. Raikwar et al.,2008)

Primary sources of Heavy metals

Pb Battery plant, Refinery, Smelter, Fuel combustion, Leaded gasoline, Lead-based paints, Lead-soldered food cans, Lead plumbing pipes & automobile exhaust (Tetraethyl lead) (McGraw-Hill, 2006)

Cd Tannery, smelter, battery crushing unit, mining, Electroplating, Pigments (Cd yellow) and plastics

(McGraw-Hill, 2006)

As Pesticides, Wood preservative, Glass/Copper smelters, Coal combustion & Uranium mining.

(H.S.Sandhu, 2nd ed, 2012)

Hg Refinery, Plastic, Paints, Antiseptic, Scientific instruments, Photography, Fuel combustion. (H.S.Sandhu,2nd ed, 2012)

Secondary sources of heavy metals

Most of the animals affected Industrial & Domestic wastage directly/indirectly release in water (Pb, Hg, Cd, As) (Bell et al., 2001)

Contd...

Agriculture soil contaminated by Heavy metals through…

Long-term use of phosphatic fertilizers (Cd) Sewage/sludge application (Hg, Cd) Dust from smelters, industrial waste (Pb, Hg, Cd, As) Bad watering practices in agricultural lands (Pb, Hg, Cd, As) (Bell et al., 2001)

Plants are contaminated by Heavy metals through…

Excessive use of fertilizers/pesticides/insecticides Plants growing in soil contaminated area Irrigation of crop by contaminated water

Grazing in contaminated

area

Crops growing in contaminated

soil

Drinking Contaminated

water

Toxicological compounds

Excessive use fertilizers & chemicals

Use of some drugs that predispose Metal toxicity

HEAVY METALS IN AYURVEDIC MEDICINES Karela tablets, produced by Shriji Herbal Products, India Karela capsules, produced by Himalaya Drug Co, India Karela capsules, produced by Charantia, UK (specifically

batch #12011) Maha Sudarshan Churna powder, produced by Zandu

Pharmaceuticals, Mumbai, India Maha Sudarshan Churna powder, D & K Pharmacy,

Bhavnagar, India Maha Sudarshan Churna powder, produced by Chhatrisha,

Lalpur, India Maha Sudarshan Churna powder, produced by Dabur India

Ltd, New Delhi, India SAFI liquid, produced by Hamdard-WAKF-Pakistan SAFI liquid, produced by Hamdard-WAKF-India Yograj Guggul tablets, produced by Zandu Pharmaceuticals,

Mumbai, India Sudarshan tablets, produced by Zandu Pharmaceuticals,

Mumbai, India Shilajit capsules, produced by Dabur India Ltd, New Delhi,

India (WHO Drug Information Vol. 19, No. 3, 2005).

E-WASTE

Pollutants

Occurrence

Arsenic Semiconductors, diodes, microwaves, LEDs (Light-emitting diodes), solar cells

Cadmium Batteries, pigments, solder, alloys, circuit boards, computer batteries, monitor cathode ray tubes (CRTs)

Lead Lead rechargeable batteries, solar, transistors, lithium batteries, PVC (polyvinyl chloride) stabilizers, lasers, LEDs, thermoelectric elements, circuit boards

Mercury Components in copper machines and steam irons; batteries in clocks and pocket calculators, switches, LCDs

Pollutants and their occurrence in waste electrical and electronic equipment

(P. Srisudha, ‘Tackling e-waste’, The Hindu, 28 June, 2009)

Soil Water Air

Primary source

Plants

Domestic animals

(Brady, 1994))

Metal Livestock's drinking Water (µg/ml)

Irrigation water (µg/ml)

Soil (µg/gm)

Plant (µg/gm)

Pb 0.10 0.06 100 0.30

Hg 0.01 0.01 30 0.O3

Cd 0.05 0.01 3 0.10

As 0.2 0.01 20 0.1

Indian standards (Awashthi, 2000) & WHO, 1999, 2011, FAO, Chiroma et al; 2014, Schütze and Throl (2000)

Maximum permissible limits of heavy metals

How its affecting food chain: bioaccumulation & biomagnification

Lead

Cattle, Horse, Dog more susceptible Pig & Cat rare (H.S.Sandhu 2nd ed, 2012)

Younger Vs. old Ruminants Vs. Non ruminants (Neathery, 1984)

Pb-acetate Pb-oxide Pb-carbonate (H.S.Sandhu 2nd ed, 2012)

Ubiquitous environmental contaminant. (H.S.Sandhu 2nd ed, 2012)

Affected organs/system

Pb

Neuro-T

GIT-T

Hepato-T

Nephro-T

Endocrine-T

Reproductive-T

Mechanism underlying the development of oxidative stress in a cell on lead exposure (Gagan flora at al.,2012)

Under normal physiological conditions, there is a balance between free radicals and antioxidants and any deviation from it can cause oxidative stress leading to cell death. (Gagan flora at al.,2012)

Cattle : show head pressing behaviour.

LEAD POISONING: CLINICAL SIGNS

Cattle: advanced stages of lead poisoning, become frenzied, bellow, stagger and crash into obstacles

Gastrointestinal signs include colic, constipation for several days followed by diarrhoea.

Abortion(mid or late gestation), opisthotonos, salivation, lacrimation and paralysis may also be observed.

Death may occur within several hours or days. (O.M.Radostits et al. 10th Ed.)

Mercury (Hg)

Minamata(1953--60) & Niigata(1964--65) disease in Japan (Mottet et al, 1985)

Elemental-Hg – non toxic (orally), highly toxic(inhalation) Inorganic-Hg – less toxic (insoluble < soluble) Organic-Hg – more toxic (H.S.Sandhu,2nd ed,2012)

Liquid forms at room temp Young ruminants more susceptible than Horse & Pig (H.S.Sandhu,2nd ed,2012)

Se & Vitamin E protects against toxicity (Parizek et al., 1974)

Structural and functional disintegration of the enzymes (–SH group) ( Roy Chowdhury A and Vachhzajani KD; 1987)

Mercury (Hg)

Hg

Lung

GIT

Skin

Absorption

Organic-Hg

Inorganic-Hg

Deposition

Bile & Faeces

Urine Hairs & others

Elemental-Hg

Faeces

Elimination

The intestinal uptake and subsequent distribution of organic mercurials, such as methylmercury, throughout the body. a. Conjugation with glutathione (GSH), shown as CH3—Hg—SG. b. Secretion of conjugate into bile. c. Reabsorption in gallbladder. d. Remaining Hg enters intestinal tract.

The ability of organic mercurials to cross the blood–brain barrier and the placentacontributes to their greater neurological and teratogenic effects when compared with inorganic mercury salts. Note the structural similarity of the methylmercury complex to methionine, CH3SCH2CH2—CH(NH3 +)COO–.

Affected organs/system

Hg

Neuro-T

GIT-T

--SH

Nephro-T

Endocrine

Repro-T

Mercurial salts

stomatitis,pharyngitis,vomiting,diarrhea

,dehydration, and shock.Death may occur within hours.

Oliguria and azotemia, lasting for 1-2 days, follow in animal animals that survive acute

mercuric ion toxicosis

Mercury Toxicity: Clinical Signs

(O.M.Radostits et. al.,10th ed.)

Regulatory limit in agricultural soil is 100 mg/kg soil. (Salt et al., 1995)

Itai Itai disease

> 5ppm toxic effect

Most common in Ruminants (NRC,1980)

CADMIUM (Cd)

Antagonistic activity against Cu, Zn, Se & Fe (chemical similarities & competition for binding ) (Ammerman et al., 1973) Oxidative stress

Destroy the SOD (Cd replaces Zn2+ ) (Zn maintain the str. of SOD that scavenges the FR) (Darbre, 2006) Inhibits the GSHB-Px (catalyzed the destruction of H2O2 & LP & protects the lipids membrane from peroxide damage)

Cd involved in Metal interaction

Affected organ/system

• Anemia• Retarded growth

• Proteinuria• Glycosuria,• Hyperphosphatemia

• Testicular degeneration and necrosis

• Arthropathy and osteoporosis

• Vomition and diarrhoea in acute cases.

(O.M.Radostits et al. 10th Ed.)

Clinical Signs

More abundance in the Earth’s crust 1.5–3.0 mg/kg (20th most abundant element) (Mandal and Suzuki, 2002)

Used as first drug to cure syphilis by Paul Erlich (Waxman and Anderson, 2001)

Most extensive exposure through drinking water

In Bangladesh 1980, arsenic-contaminated Artesian well water. (Mandal and Suzuki, 2002)

Oxidative stress

Carcinogenicity

Arsenic

(Casarett and Douls,7th ed.)

SUBACUTE: Bloody diarrhoea & dehydration.Weakness and hind limb paralysis

Organic Arsenic: Blindness and incordination mainly occur in overdosing of arsenilic acid.In swine dog sitting posture

ARSENIC TOXICITY CLINICAL SIGNS

Chronic:Low body weight & sloughing of skin

(O.M.Radostits et al. 10th Ed.)

Management of Heavy Metal Toxicity

1. Decontamination• Removal of the patient from the source of exposure is critical

to limiting dose.• Emetics, activated charcoal, gastric lavage employed if

ingestion is recent.• Charcoal administered @1-4 mg/kg P/O.

2. ResuscitationGood supportive care is critical.

3. Chelation

Basic principles of metal toxicity management :(1) Prevention of further metal absorption into the system (2) Elimination of metal from the circulation (3) Inactivation of metal bioavailable in the system

• Chelation has its origin in the Greek word chele that means claw of a lobster, thus depicting the concept of clinging or holding with a strong grip.

• The term chelate was first applied by Sir Gilbert T. Morgan and H. D. K. Drew in 1920.

They suggested the term for the caliper-like groups which function as two associating units and fasten to a central atom so as to produce heterocyclic rings

(T. Morgan et. al.,1920)

CHELATION

(O.Andersen,1999)

(Swaran J.S. Flora and Vidhu Pachauri , 2010)

Edetate Calcium Disodium

Treatment of poisoning by metals that have higher affinity for the chelating agent than does ca2+.

EDTA is charged at physiological pH, it does not significantly penetrate cells; its volume of distribution approximates extracellular fluid space.

Lead Poisoning.• Bone provides the primary source of lead that is chelated by

CaNa2EDTA• After such chelation, lead is redistributed from soft tissues to the

skeleton• Calcium versenate (Ca Na2 EDTA, Ca EDTA) @ 110-220 mg/kg

BW IV infusion, 2 times a day (as 1-2% solution in 5% dextrose) for 4-5 days ( Large animals)

(CALCIUM DISODIUM VERSENATE)

• Mercury is unavailable to the chelate perhaps because it is too tightly bound by sulfhydryl groups or sequestered in body compartments that are not penetrated by CaNa2EDTA.

Toxicity: hypocalcemic tetany, hydropic vacuolization of the proximal tubule, loss of the brush border, and eventually, degeneration of proximal tubular cells (Catsch and Harmuth- Hoene, 1979).

• Like EDTA, is a polycarboxylic acid chelator, but it has somewhat greater affinity for most heavy metals.

Pentetic Acid (DTPA/ Di ethylene tri amine penta acetic acid)

• Limited access to intracellular sites of metal storage

• Because DTPA rapidly binds ca2+, CaNa3DTPA is employed

Heavy-metal poisoning that do not respond to EDTA, particularly poisoning by radioactive metals like Uranium and Plutonium

(N.L.Spoor, 1977)

Disadvantage of depleting zn from the system that may be overcome by supplementation or using the zinc salt of the drug.

Teratogenic like CaNa2EDTA due to its Zn and Mn depletion effect

Developed during world war II as an antidote to lewisite, a vesicant arsenical war gas, hence its alternative name, british antilewisite (BAL).

Dimercaprol(2,3dimercaptopropanol)

Its instability in aqueous solutions, peanut oil is the solvent employed in pharmaceutical preparations.

Arsenicals would form a very stable and relatively nontoxic chelate ring with the dimercaprol

MOA: Formation of chelation complexes Between its sulfhydryl groups and metals• Antagonizes the biological actions of metals that form mercaptides

with essential cellular sulfhydryl groups, principally arsenic, gold, and mercury.The sulfur–metal bond may be labile in the acidic tubular urine,

which may increaseDelivery of metal to renal tissue and increase toxicity.Maintain a concentration of dimercaprol in plasma adequate to favor the continuous formation of the more stable 2:1 (BAL–metal) complex and its rapid excretion

More effective in preventing inhibition of sulfhydryl enzymes than in reactivating them.

Used in combination with CaNa2EDTA to treat lead poisoning, especially when evidence of lead encephalopathy exists.

Dimercaprol cannot be administered orally; it is given by deep intramuscular injection as a 100 mg/ml solution in peanut oil,

Toxicity: rise in systolic and diastolic arterial pressures, accompanied by tachycardia

Arsenic toxicity: BAL(British Anti-lewisite)/Dimecaprol:@4-7mg/kg I/M t.i.d×3days.

An orally effective chelator that is chemically similar to dimercaprol but contains two carboxylic acids that modify both the distribution and chelating spectrum of the drug.

Succimer (2,3-dimercaptosuccinic acid, CHEMET)

After Absorption

Effective as a chelator of arsenic, cadmium, mercury, and other metals

(Aposhian and Aposhian, 1990)

Toxicity : less than that with dimercaprol perhaps because its relatively lower lipid solubility minimizes its uptake into cells

A desirable feature : it does not significantly mobilize essential metals such as zinc, copper, or iron.

First isolated in 1953 from the urine of patients with liver disease who were receiving penicillin

Penicillamine (D-β,β-dimethylcysteine)

Effective chelator of copper, mercury, zinc, and lead and promotes the excretion of these metals in the urine.

Absorbed (40% to 70%) from the GI tract

N-Acetylpenicillamine is more effective than penicillamine in protecting against the toxic effects of mercury presumably because it is even more resistant to metabolism.

Toxicity. With long-term use, induces several cutaneous lesions, including urticaria, macular or papular reactions, pemphigoid lesions, lupus erythematosus, dermatomyositis, adverse effects on collagen,

Chelation

MonotherapyCombination

Therapy

AntioxidentsMicronutrientsPhytochemicals

Acute Metal Exposure

Soft Tissues

Cellular ManifestationPro – vs antioxident imbalance Metabolic

pathway interfered (haem synthetic pathway)

Tissue Damage & Organ dysfunction

Cellular ManifestationOxidative Stress, Pro- or Anti-

apoptotic manifestations (Mitochondrial dysfunction,

DNA damage, etc)

Systemic ManifestationsDisease induction or

promotions(Diabetes,Cancer, DVD, etc)

Soft & Hard Tissues

Chronic Metal Exposure

Excretion (Urinary / Biliary)

(Swaran J.S. Flora and Vidhu Pachauri , 2010)

• Greater Affinity, Low Toxicity

•Ability to compete with natural chelators •Ability to penetrate cell membranes

•Rapid elimination of the toxic metal

•High water solubility

•Capacity to form non-toxic complexes

•Same distribution as the metal

(Swaran J.S. Flora and Vidhu Pachauri , 2010)

IDEAL CHELATER

Benefits • Effective against

acute poisoning • Form non-toxic

complexes • Remove metal

from soft tissues • Oral therapy is

available

Drawbacks• Redistribution of toxic

metal • Essential metal loss • No removal of metal from

intracellular sites • Hepatotoxicity and

nephrotoxicity • Poor clinical recovery • Pro-oxidant effects (DTPA) • Headache, nausea,

increased blood pressure

CHELATION

PREVENTION AND CONTROL

BIOREMEDIATION

Use of different biological systems to destroy or reduce concentrations of contaminants from polluted sites.

Microbes and plants have a natural capability to attenuate or reduce: Mass,Toxicity, Volume, Concentration of pollutants

Aerobic bacteria:Examples include: Pseudomonas, Alcaligenes, Sphingomonas, Rhodococcus, and Mycobacterium.

Fungi:Able to degrade a diverse range of persistent or toxic environmental pollutants

(Bodishbaugh, D.F., 2006)

Phytoremediation is the use of living green plants for in situ risk reduction and/or removal of contaminants from contaminated soil, water, sediments, and airHyper accumulator plant species are used on many sites due to their tolerance of relatively extreme levels of pollution.Avena sp. , Brassica sp.

BIOREMEDIATION

Use of different biological systems to destroy or reduce concentrations of contaminants from polluted sites.

Microbes and plants have a natural capability to attenuate or reduce: Mass,Toxicity, Volume, Concentration of pollutants

Aerobic bacteria:Examples include: Pseudomonas, Alcaligenes, Sphingomonas, Rhodococcus, and Mycobacterium.

Fungi:Able to degrade a diverse range of persistent or toxic environmental pollutants

(Bodishbaugh, D.F., 2006)

Phytoremediation is the use of living green plants for in situ risk reduction and/or removal of contaminants from contaminated soil, water, sediments, and airHyper accumulator plant species are used on many sites due to their tolerance of relatively extreme levels of pollution.Avena sp. , Brassica sp.

68

PHYTOREMEDIATION

Phytoextraction

1

Phytovolatilization

2

Phytostabilization

3

Rhizodegradation

Rhizofiltration

4

5

5 mechanisms based on the fate of contaminants